Nu-ethyloxytetracycline



United States Patent 3,394,178 N-ETHYLOXYTETRACYCLINE Eugene L. Dulaney, Summit, and Irving Putter, Martinsville, N.J., assignors to Merck & Co., Inc, Rahway, N.J., a corporation of New Jersey No Drawing. Continuation-impart of application Ser. No. 185,233, Apr. 5, 1962. This application Jan. 26, 1965, Ser. No. 428,217

Claims. (Cl. 260-559) This application is a continuation-in-part of our pending application Ser. No. 185,233, filed Apr. 5, 1962, now abandoned.

This invention relates to new tetracyclines and processes of preparing the same. More particularly, it is concerned with the preparation of new tetracyclines wherein one of the N-methyl substituents is an ethyl substituent.

The tetracycline group of antibiotics such as tetracycline, chlortetracycline and oxytetracycline have been found to be very valuable broad spectrum antibiotics. In addition, certain derivatives of these tetracyclines such as the 6-demethyl tetracyclines have also been found to be valuable antibiotics. All of these previously known antibiotics are characterized by having .a dimethylamino substituent at position 4.

It is an object of the present invention to provide new tetracycline antibiotics having a methyl ethyl amino substituent at position 4. An additional object is to provide processes for preparing tetracycline antibiotics having a methyl ethyl amine substituent at position 4. Other objects will be apparent from the detailed description of our invention hereinafter provided.

In accordance with the invention, it is now found that tetracyclines having an N-ethyl methyl substituent at the 4 position are obtained by growing a tetracycline producing microorganism in aqueous mediums in the presence of ethionine. Thus, pursuant to a specific embodiment of the present invention, N-ethyloxytetracycline, or 4-ethylmethylamino 1,4,4a,5,5a,6,11,12a octahydro 3,5,6, 10,12,12a hexahydroxy 6 methyl 1,11-dioxo-2- naphthacenecarboxarnide, is produced by growing an oxytetracycline producing strain of microorganism in the presence of a minor amount of ethionine. The structure of the Nethyl-oxytetracycline produced can be represented as follows:

CH3 OH O HO 0 OH ii Thus, N-ethyloxytetracycline is produced together with oxytetracycline with Streptomyces rimosus is grown in suitable fermentation mediums containing a minor amount of ethionine. After the completion of the fermentation, the mixed antibiotics are recovered from the fermentation broth, and the desired N-ethyloxytetracycline separated from said mixture.

The amount of ethionine in the fermentation medium will depend upon a particular oxytetracycline producing microorganism and the amount of methionine present in the medium constituents and synthesized by the organism. It is found that mediums containing more methionine will require larger amounts of ethionine. In general, it is necessary that the medium contain at least 5 ug. of ethionine per m1. of medium in order to produce the N-ethyloxytetracycline. The optimum :amount needed for a particular microorganism can readily be determined experimentally by carrying out a few fermentations with mediums containing various amounts of ethionine. The ethionine used to supplement the fermentation mediums can be d-thionine, l'ethionine, dl-ethionine or mixtures thereof.

It is found that various strains of Streptomyces rimosus and =auxotrophs thereof prepared by mutation are satisfactory in producing N-ethyloxytetracycline by fermentation in the presence of ethionine. Thus, the strain of Streptomyces rimosus deposited in the culture collection of the Fermentation Division of the Northern Regional Research Laboratories at Peoria, 111., as NRRL 2234 can be grown in the presence of ethionine to produce the new antibiotic of the present invention.

Pursuant to another specific embodiment of this invention, it is found that certain methionine dependent auxotrophs of Streptomyces rimosus obtained by ultraviolet irradiation in accordance with procedures known in this art are especially useful in producing N-ethyloxytetracycline. One such auxotroph which is identified in the culture collection of Merck & 00., Inc. at R-ahway, N.J., as MA 935 has been deposited in the culture collection of the Northern Regional Research Laboratories where it is identified as NRRL 2970.

The following examples are presented to illustrate methods of producing the new tetracycline of the present invention.

Example 1 A methionine dependent auxotroph of Streptomwces rfmosus NRRL 2970 is grown as follows:

Inoculum is developed for three days at 28 C. on rotary shakers moving at 220 rpm. in 50 ml. of the following medium in 250 ml. Erlenmeyer flasks:

Distilled water to 1 liter.

The resulting growth is washed twice with 0.85% NaCl solution and made up to volume in fresh saline. Two and one-half ml. of washed growth is used to inoculate 50 m1. of production medium in a 250 ml. flask. The production medium is made as follows:

Sucrose g 40 Cottonseed endosperm flour g 8 4)2 4 6 CaCO g 11 ZnSO .7H O mg 30 Distilled water at 1 liter.

Replicate flasks also receive 200 g/ml. of l-methionine and others 100 ,ug/ml. of dl-ethionine.

The inoculated production flasks are incubated at 28 C. on rotary shakers moving at 200 rpm. for hours. They are then assayed for total antibiotic productivity using a cylinder plate agar diffusion assay.

Extracts of the broths are made for chromatography as follows:

The broth is acidified to pH 1-2 with HCl, filtered through paper and the filtrate extracted twice with equal volumes of n-butanol. The n-butanol extracts are combined, the n-butanol removed by lyophilization and the residue taken up in two ml. of pure methanol. The methanol extracts are used for spotting paper chromatograms.

MacIlvaines buffer at pH 4.7 is used to wet the paper prior to applying the extracts. Ethyl acetate is used as the mobile phase. After development, the papers are dried, treated with ammonia vapors and examined with ultraviolet light. The U.V. fluorescing areas are marked with a pencil and the paper chromatograms laid on a plate seeded with Escherichia coli. After incubation overnight, the :bioactive areas are noted. In each of the fermentations carried out with the auxotroph, oxytetracycline is found to be the major antibiotic in the broths. However, a more mobile, bioactive product is also present in the cultures. This more mobile product is N-ethyloxytetracycline.

Example 2 Fermentation broth, prepared as described in Example 1, is acidified with hydrochloric acid to pH 1.52.0 and filtered. To the filtrate is added 25% by Weight of sodium chloride. The resulting solution is extracted with n-butanol using multiple extractions. The butanol layers ml. of water at pH 1.52.0. The water extracts are adjusted to pH 9. After adding 45 g. of sodium chloride, the solution is extracted three times with 180 ml. of butanol. The butanol solution is adjusted to pH 3 and concentrated to 30 ml. and the tetracycline extracted into 230 ml. portions of water at pH 1.5. The water extract is concentrated to dryness at low temperature and the residue dissolved in 5 ml. of methanol. This solution contained bioactivity equivalent to 3 mg. of oxytetracycline.

Four Whatman 3 mm. filter paper sheets 7" wide were Wet with pH 4.5 MacIlvaine buffer and the concentrate streaked evenly on the four sheets. The chromatogram is developed for four hours and the zones visualized by inspection of the sheets with ultraviolet light. The irregular zone containing the N-ethyloxytetracycline is cut out and slurried with pH 4.5 phosphate buffer. The buffer eluate contains 100' of bioactivity as oxytetracycline and gives a typical oxytetracycline ultraviolet absorption spectrum.

Example 3 Twenty gallons of fermentation broth produced as described in Example 1 is extracted into butanol and back into water. The water solution, 12 liters, is concentrated further by the following procedures:

To the 12 l. of concentrate is added 5 liters of ethyl acetate, 250 g. of sodium barbital and g. of calcium chloride. The pH is adjusted to 8.89.2 and the phases separated. The solvent phase is Washed with one liter portion of water containing g. of sodium barbital and 10 g. of calcium chloride and four 10 liter portions of water saturated with ethyl acetate. Most of the oxytetracycline is discarded in the washes. The residual ethyl acetate is diluted with an equal volume of light petroleum ether and extracted 5 times with 0.1 N hydrochloric acid. These extracts are combined into two fractions and freezedried yielding the following products:

Fraction I, 670 mg., Biopotency 10.5 y/mg. II, 2.2 g., Biopotency 18 t/mg.

300 mg. from fraction I is dissolved in methanol, precipitated with ether, redissolved in methanol and streaked on 4" x 7" sheets of Whatman 3 mm. paper. Elution of the separated zone gives 500'y of single spot material by ultraviolet absorption. Similar treatment with 1.5 g. of the 2.2 g. fraction yielded 250 of N-ethyloxytetracycline.

4 Example 4 An aqueous medium containing the following nutrients Percent Soluble starch 3 Dextrose 0.2 (NH SO 0.5 CaCO 0.5 NaCl 0.5 K HPO 0.2 MgSO .7H O 0.1

FeSO .7H O 0.00 ZnSO .7H O 0.001 dl-Methionine 0.02

is prepared and 50 ml. of the medium in 250 ml. flasks is sterilized by heating at C. for fifteen minutes.

The sterilized medium is inoculated with the growth from an agar slant of culture NRRL 2970 suspended in 10 ml. of the sterile medium. The flask is incubated for three days on a rotary shaker at 28 C.

At the end of this incubation period, 10 ml. of the broth is used to inoculate 400 ml. of the above-described sterile medium in a 2-liter Erlenmeyer flask. The inoculated medium is then inoculated for three days at 28 C.

The resulting broth is used to inoculate about 40 gallons of the above-described sterile medium in a fifty gallon stainless steel fermentor. The fermentation is then allowed to proceed with aeration and agitation for 48 hours at 28 C. About 43 liters of the fermented broth so obtained is used to inoculate 120 gallons of the sterile production medium having the following composition:

Percent Cottonseed endosperm flour 2 DL-ethionine 0.01 Glycerol 4 ZnSO, 0.00 3 CaCO 1 pH before sterilization 6.7.

The fermentation is allowed to proceed with aeration and agitation for 120 hours at 28 C.

Samples of the broth are analyzed by paper strip chromatography and bioautography. The broths Were found to contain oxytetracycline and N-ethyloxytetracycline. Based on this method of assay, using a chlortetracycline standard, the broth is found to contain approximately 8 ug/ml. of the N-ethyloxytetracycline at the end of the fermentation period.

Example 5 A fermentation is carried out as described in Example 4 using culture NRRL 2970, a methionine dependent auxotroph.

After completion of the fermentation, the 110 gallons of broth is adjusted to pH 1.5 with hydrochloric acid and filtered. To the filtered broth is added g. of sodium chloride per liter of broth, and the broth extracted three times with one fifth volume of n-butanol. The solvent extracts are combined and washed twice with one-tenth volume of 15% sodium chloride at pH 9. The butanol layer is evaporated in the presence of water at pH 2.5 to an aqueous concentrate having a volume of about four liters.

To the four liters of concentrate is added 1.86 liters of a water solution containing 360 g. of BaCl .2H O and 48g. MgCl .6H O per liter of water, and the solution ad usted to pH 8.5 with sodium hydroxide. The recipitate which forms is recovered by centrifugation and then slurried with 280 ml. of water. The slurry is adjusted to pH 1.5 with sulfuric acid and the barium sulfate separated and Washed with water. The acid soluble portion 1s adjusted to pH 7 thereby precipitating the tetracycline antibiotics as the barium-magnesium salts. The precipitate is recovered and dissolved in methanol. This solution is found to contain the equivalent of 4.7 g. of tetracyclines as determined by the U.V. measurement at 355 m Paper chromatography indicated that about 7% of the Example 7 total tetracyclines is N-ethyloxytetracycline.

The methanol solution is chromatographed on 3 mm. paper using the partition system ethyl acetate-pH 4.7 phosphate buffer (0.05 M). The spot on the paper corresponding to N-ethyloxytetracycline, as seen by fluorescence, is 353 m Ellicut out of the Paper and the activity eluted with N is treated with 1.5 ml. of an aqueous solution of barium hydrochloric acid. The acidic solution so obtained is found and magnesium Salts (36 o+ g to contain 73 mg. of N-ethyloxytetracycline as determined MgCi2 6H2O per 100 mis and the mixture adjusted m by UV. absorption. The antibiotic is extracted into n- PH 85 with sodium hydroxide The barium magnesium butanol and the solvent evaporated to about 20 ml. This Sah of N eihyioxytetracyciihe which iS precipitated iS concentrate is then sub ected to a 20 plate extended mul- Separated by centrifugation, washed with water, iyophh tiple extraction using the partition system 0.01 N HCl vs. iized and dried at c f 6 hOurs in Vacuu The Solid butanol. In this system 20 ml. of butanol equilibrated with Obtained weighed 40 mg lower phase was placed in 20 extraction vessels and then The ultraviolet absorption spectrum oi the dried barium twenty ml. portions of 0.01 N hydrochloric acid conmagnesium salt is found to have peaks at 270 m, and 353 tacted with each of the butanol phases in such a way that which are identical to the position of the peaks for water phase No. 1 1s extracted successively with each oxytetracyciine in 001 N Hci'The butanol phase (the last butanol is numbered 20) and then Fifty ml. of an aqueous solution containing 1.03 mg. of N-ethyloxytetracycline per ml.based on ultraviolet absorption at water phase No. 2 is treated in a similar manner, etc. 20 At the completion of the operation, the N-ethyloxytetraof the barium magnesium salt is found to be 268 at 353 cycline is obtained by combining butanol phases 1420 m and water phases 13 through 20. The solutions are evapo- The infrared spectrum of the barium magnesium salt rated into butanol at pH 3.3. Butanol insoluble salts are of N-ethyloxytetracycline is indistinguishable from that filtered off and the rich butanol filtrate is evaporated into of oxytetracycline. water to yield a solution containing one mg. per ml. of A solution containing 100 of N-ethyloxytetracycline Naethyloxytgtracycline is assayed in triplicate by agar plate diffusion with the stamp procedure on concentrations of 40 ,ug./ml. and 10 Example '6 ng/ml. The results of these tests run in comparison with samples of oxytetracycline ig/ml. and 10 lg/ml.) Following the procedure described in Example LStrepand tetracycline (20 ngJml. and 5 ,agJml.) are shown tomyces rimosus was grown in a medium containing in the following table;

ANTIBACTERIAL SPECTRUM OF N-EIHYLOXYTETRACYCLINE Average Inhibition Zone Diameter, mm.

Test Organism N-ethyloxy- Oxy- Tetracycline N-ethyloxy- Oxy-- Tetracycline tetracychne tetracycline 20 g/ml. tetracycline tetracycline 5 #gl/ml.

40 pgjml. 40 g/ml. 10 rgJml. 10 gJml.

Escherichia coli W (MB-60) (ATCC 9637) 24 23 19 17 18 14 Bacillus sp. (M13433) 39 36 35 31 31 26 Proteus vulgan's (MB-1012) 16 16 11 14 14 0 Pseudomonas acruginosa (MB-979) 22 19 16 14 15 0 Serratia marcescens (MB'252) (ATGO 990) 0 0 0 0 0 [J Staphylococcus aureus (MB-108) 2091 42 38 41 36 36 33 Bacillus subtilis (MB-964) (ATCC 6633) 40 56 34 31 33 29 Surcina l'utea (MB-11011 (ATCC 9341). 37 32 35 27 27 27 Staphylococcus aureus, Streptomycin Resistant (MB- 0 698 31 33 32 26 28 28 Slreptoccocusfaecalis (MB-753) 1001 21 22 21 19 23 17 Alcaligcnesfaecalis (MB-l0) (A'ICO 213) 21 19 16 12 7 O Brucelln bronchiseptica (MB-965) (ATCC 4617). 38 30 30 29 22 23 Salmonella gallinarum (MB-1287) 26 25 19 19 20 13 Vibrio percolans (MB-1272) (ATCC) 8641 36 32 29 29 29 24 X anthomonas vesz'catoria (MB-815) 27 27 23 21 22 17 microcuries of l-ethionine 1n Wh1ch the ethyl group is N-ethyloxytetracychne solution 1s also assayed versus C labeled on carbon 1. After the completion of the E. coli by the stamp method with agar plates buffered fermentation, the labeled tetracycline is isolated by paper with 0.1 M phosphate at pH 5 and 9. The results of these strip chromatography following the procedures described assays compared to results obtained with oxytetracycline, in Example 2. The radio active tetracycline was found to tetracycline and chlortetracycline are shown in the folbe a single component material when chromatographed in lowing table:

0.1 M phosphate N-ethyloxy- Oxytetra- Tetra- Ohlortetrabuttered agar cycline cycline cycline cycline 40 kg./m1. 4O leg/m 20 lag/ml. 20 lag/ml.

pH 26 25 19 23 pH 27 2e 23 16 several solvent systems with the UV. and radio activity The paper chromatograms of N-ethyloxytetracycline coinciding. were compared with five known tetracyclines in the fol- Alkaline hydrolysis of the radio active tetracycline lowing partition systems by descending chromatography: showed that all of the radio activity in the sample was F acetate, Macnvaine buffer PH 4.5 (Stationary associated with the volatile amine which was liberated on 1 phase) degradation. The amine so liberated was identified using H Nitromethahe chiomfoi-m pyridihe 204 4 (H M the chromatographic method as methyl ethyl amine. This pH 3 phosphate buffer (stationary phase) analysis was also confirmed y NMR p py- IlIButanol-chloroform (9-1), 0.3 M phosphoric acid-- This experiment showed the unknown to be N-ethyloxy- 7f) 01% t i hl a ti id H 2 tetracycline. IV-Butanol, 0.1 M pH 3 phosphate buffer The movement of the tetracyclines is tabulated in mm. in the following table:

N01E.-OXT Oxytetracycline; OTC Chlortetracycline; TC =Tetracycline; and NEO =N-ethyloxytetracyclinc.

The N-ethyloxytetracycline has the same mobility as chlortetracycline in system I and tetracycline in system II. However, differing mobilities in systems III and IV clearly diflferentiate the new tetracycline from any of the knowns.

The ultraviolet light absorption spectrum of N-ethyloxytetracycline characterizes it as a tetracycline.

Stability in acid and alkali is characteristic of the tetracyclines. Since the first step in the degradation of tetracyclines having a tertiary hydroxyl at C is dehydration of this hydroxyl, marked stabilization is obtained by either removing the hydroxyl (6-deoxy series) or the methyl (6-demethyl series). These derivatives are quite stable even in 0.1 N acid or alkali. Rapid destruction of the ultraviolet absorption on treatment with 0.1 N alkali indicates that the new tetracycline has a tertiary hydroxyl at C When the new tetracycline is heated overnight at 60 C. in 0.5 N HCl, a new substance having ultraviolet absorption peaks at 248 and 370 is produced. These peaks are characteristic of apooxytetracycline' and indicate the pres ence of a S-hydroxy group.

N-ethyloxytetracycline is an amphoteric compound which forms salts on reaction with either pharmaceutically acceptable inorganic acids or with alkali or alkaline earth metal hydroxides. The term pharmaceutically acceptable inorganic acids designates inorganic acids ordinarily employed in the production of acid salts of biologically active bases suitable for pharmaceutical use although the acids themselves are not ordinarily used as pharmaceuticals except in very dilute solution. The pharmaceutically acceptable inorganic acid salts formed by reaction with the pharmaceutically acceptable inorganic acids are acid addition salts of N-ethyloxytetracycline such as are formed by the reaction of N-ethyloxytetracycline with phosphoric acid, nitric acid, sulfuric acid, hydrochloric acid and hydrobromic acid. The acid addition salt and the alkali metal, e.g. sodium and potassium, and alkaline earth metal, e.g., calcium and magnesium salts of N-ethyloxytetracycline are prepared in the same manner as the corresponding salts of the known tetracyclines such as chlortetracycline, oxytetracycline and tetracycline. Thus, these salts are prepared by reacting an equivalent of N- ethyloxytetracycline and an acid or a base in water or a lower alkanol such as methanol and subsequently recovering the formed salt of N-ethyloxytetracycline by evaporating the water or organic solvent from the reaction solution to obtain the desired salt in solid form. In this manner, by mixing N-ethyloxytetracycline with equivalent amounts of phosphoric acid, nitric acid, sulfuric acid, hydrochloric acid or hydrobromic acid, there is formed, respectively, the phosphate salt, the nitrate salt, the sulfate salt, the hydrochloride salt, or the hydrobromide salt of N-ethyloxytetracycline. In similar manner, by reaction of N- ethyloxyltetracycline with equivalent amounts of sodium hydroxide, potassium hydroxide, magnesium hydroxide or calcium hydroxide in methanol solution, there is formed, respectively, the sodium salt, the potassium salt, the magnesium salt or the calcium salt of N-ethyloxytetracycline, each of which may be recovered in solid form by evaporation of the methanol from the solution of formed salts. The salts of N-ethyloxytetracycline possess antimicrobial activity which varies slightly from one to another, depending on the proportion by weight of antibiotic in the salt.

In vivo tests, using four-fold dilution increments of drug with five mice at each level, show the N-ethyloxytetracycline to be less active than oxytetracycline when used intraperitoneally against infection with Streptococcus pyogerzes C203 or Salmonella sclzoltmuelleri MI, and intraperitoneally or subcutaneously against infection with Staphylococcus aurcus Smith. However, it was observed to be about twice as active as oxytetracycline when used intraperitoneally against infection with Proteus vulgar-1's l8l0.

The new antibiotic and its acid and metal salts of this invention are valuable bactericidal substances which are active against various gram-positive and gram-negalive microorganisms. As indicated above, they are similar in activity to the other tetracycline antibiotics and hence useful for many of the same purposes as these products, for example, as therapeutic agents. It can also be used as a germicidal agent, for example, for the sterilization of pharmaceutical equipment and the like. N-ethyloxytetracycline is useful in the isolation of microorganisms from soil samples. Thus, by adding a small amount of the antibiotic or a salt thereof to the agar medium upon which the soil samples are plated, the growth of many of the organisms contained in the soil is inhibited. This inhibition of the growth of many of the bacteria facilitates the recovery of individual colonies of microorganisms from the plated agar.

Various changes and modifications of the invention can be made, and to the extent that such variations incorporate the spirit of this invention, they are intended to be included within the scope of the appended claims.

What is claimed is:

1. A compound selected from the group consisting of N-ethyloxytetracycline, pharmaceutically acceptable inorganic acid salts and alkali and alkaline earth metal salts of N-ethyloxytetracycline.

2. N-ethyloxytetracycline hydrochloride.

3. Alkali metal salts of N-ethyloxytetracycline.

4. Alkaline earth metal salts of N-ethyloxytetracycline.

5. A compound of the formula No references cited.

NICHOLAS S. RIZZO, Primary Examiner. 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF N-ETHYLOXYTERTRACYCLINE, PHARMACEUTICALLY ACCEPTABLE INORGANIC ACID SALTS AND ALKALI AND ALKALINE EARTH METAL SALTS OF N-ETHYLOXYTETRACYCLINE. 